This invention relates generally to enzymes that convert sucrose to isomaltulose. More particularly, the present invention relates to novel sucrose isomerases, to polynucleotides encoding these enzymes, to methods for isolating such polynucleotides and to nucleic acid constructs that express these polynucleotides. The invention also relates to cells, particularly transformed bacterial or plant cells, and to differentiated plants comprising cells, which express these polynucleotides. The invention further relates to the use of the polypeptides, polynucleotides, cells and plants of the invention for producing isomaltulose.
Isomaltulose α-D-glucopyranosyl-1,6-D-fructofuranose (also called palatinose) is a naturally occurring structural isomer of sucrose (α-D-glucosyl-1,2-D-fructose). Isomaltulose is a nutritive disaccharide, with sweetness and bulk similar to sucrose. Several characteristics make isomaltulose advantageous over sucrose for some applications in the food industry: 1) noncariogenic (not causing dental decay); 2) low glycemic index (useful for diabetics); 3) selective promotion of growth of beneficial bifidobacteria among human intestinal microflora; 4) greater stability of isomaltulose-containing foods and beverages; 5) less hygroscopic; 6) simple conversion into sugar alcohols with other useful properties as foods. The safety of isomaltulose has been comprehensively verified, resulting in unqualified approval as human food, and it is widely used commercially as a sucrose substitute in foods, soft drinks and medicines (Takazoe, 1989, Palatinose—an isomeric alternative to sucrose. In: Progress in Sweeteners (T H Grengy, ed.) pp 143-167. Elsevier, Barking, UK).
Furthermore, because isomaltulose has an accessible carbonyl group, it has attracted attention as a renewable starting material for the manufacture of bioproducts such as polymers and surfactants with potential advantages over substances manufactured from petroleum (Cartarius et al., 2001, Chemical Engineering and Technology 24: 55A-59A; Kunz, 1993, From sucrose to semisynthetical polymers. In: Carbohydrates as Organic Raw Materials II (G Descotes, ed.) pp 135-161. VCH, Weinheim; Lichtenthaler et al., 2001, Green Chemistry 3: 201-209; Schiweck et al., 1991, New developments in the use of sucrose as an industrial bulk chemical. In: Carbohydrates as Organic Raw Materials (F W Lichtenthaler, ed.) pp 57-94. VCH, Weinheim).
Commercial isomaltulose is produced from food-grade sucrose by enzymatic rearrangement from a (1,2)-fructoside to a (1,6)-fructoside followed by crystallization. Sucrose isomerase (SI) enzymes (also known as isomaltulose synthases), which are able to convert sucrose to isomaltulose, have been demonstrated in Protaminobacter rubrum, Erwinia rhapontici, E. carotovora var atroseptica, Serratia plymuthica, S. marcesens, Pseudomonas mesoacidophila, Leuconostoc mesenteroides, Klebsiella spp., Agrobacterium sp., haploid yeast and Enterobacter sp. (Avigad 1959, Biochemical Journal 73: 587-593; Börnke et al., 2001, Journal of Bacteriology 183: 2425-2430; Cheetham et al., 1982 Nature 299: 628-631; Huang et al., 1998, Journal of Industrial Microbiology & Biotechnology 21: 22-27; Lund and Waytt, 1973, Journal of General Microbiology 78: 331-3; Mattes et al., 1998, U.S. Pat. No. 5,786,140; McAllister et al., 1990, Biotechnology Letters 12: 667-672; Miyata et al., 1992, Bioscience Biotechnology and Biochemistry 56: 1680-1681; Munir et al., 1987, Carbohydrate Research 164: 477-485; Nagai et al., 1994, Bioscience Biotechnology and Biochemistry 58: 1789-1793; Nagai-Miyata et al., 1993, Bioscience Biotechnology and Biochemistry 57: 2049-2053; Park et al., 1996, Revista De Microbiology 27: 131-136; Schmidt-Berg-Lorenz and Maunch, 1964, Zeitung fur die Zuckerindustrie 14: 625-627; Stotola et al., 1956, Journal of the American Chemical Society 78: 2514-2518; Tsuyuki et al., 1992, Journal of General and Applied Microbiology 38: 483-490; Zhang et al., 2002, Applied and Environmental Microbiology 68: 2676-2682). Isomaltulose is currently produced in industrial scale column reactors containing immobilized bacterial cells. Initially, natural isolates have been used for this purpose but it is anticipated that higher yields of isomaltulose may be achieved using recombinant techniques. Mattes et al. (1998, supra) disclose isolated polynucleotides from Protaminobacter rubrum (CBS 547,77), Erwinia rhapontici (NCPPB 1578), the microorganism SZ 62 (Enterobacter species) and the microorganism MX-45 (Pseudomonas mesoacidophila FERM 11808 or FERM BP 3619) for producing recombinant partial or full-length sucrose isomerase enzymes in host cells such as Escherichia coli. Mattes et al. also disclose conserved amino acid sequences for designing degenerate oligonucleotides for cloning sucrose isomerase-encoding polynucleotides by the polymerase chain reaction (PCR).
In addition to isomaltulose, reported SIs produce varying proportions of the isomer trehalulose (1-O-α-D-glucopyranosyl-D-fructose) along with glucose and fructose as by-products. Some purified SIs produce predominantly isomaltulose (75-85%), others predominantly trehalulose (90%). The ratio of these products varies with reaction conditions, particularly temperature and pH, and under some conditions small quantities of other products such as isomaltose and isomelezitose may be formed (Véronèse and Perlot, 1999, Enzyme and Microbial Technology 24: 263-269). The formation of multiple products lowers the yield and complicates the recovery of the desired isomer. Slow conversion of sucrose into isomaltulose, and a narrow range of optimal reaction conditions also limit the industrial efficiency of isomaltulose production (Cheetham, 1984, Biochemical Journal 220: 213-220; Schiweck et al., 1990, Zuckerindustrie 115: 555-565). An ideal SI would show high speed, complete conversion, high specificity and a wide window of reaction conditions for isomaltulose production.